RESUMO
Homologous regions were revealed and analyzed within the first intron of the mouse and human mts1 genes. Maximal homology was detected between microsatellite DNA sequences from the first intron of the mouse gene (+804, +863) and the human gene (+600, +645). DNase I hypersensitive sites were revealed within these regions in both mouse and human cell lines tested, thus showing functional significance of the homology detected. In both the mouse and the human genes, the 5' end of homologous regions is flanked by a DNA sequence similar to a NF-kB/Rel protein-binding consensus sequence. Previously, this sequence was demonstrated to be involved in a complex regulatory element of the mouse mts1 gene. In comparison with the kB-like sequence, mouse, rat, and human sequences were found to involve one, two, and three nucleotide substitutions, respectively. Alterations in a spectrum of nuclear proteins bound to various kB-like sequences were analyzed. Possible effects of these alterations on mts1 transcription regulation were discussed. The possibility of the human kB-like sequence to function as positive regulator of transcription initiation was demonstrated in vivo.
Assuntos
Regulação da Expressão Gênica/fisiologia , Íntrons , Repetições de Microssatélites , NF-kappa B/genética , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Sequência Consenso , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Data on cloning of cDNA corresponding to human mts1 gene transcripts are presented. By comparing nucleotide sequences of the genomic DNA clone and cDNA of mts1, it was shown that human osteosarcoma OHS cells contain two alternative splice variants of mts1 transcripts. Alternative splicing occurs in the 5'-untranslated region of the mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1(var), demonstrate similar stability in the cells, and each contains one open reading frame for the MTS1 protein. However, the two types of transcripts are translated with different effectiveness. The level of transcription of mts1 splice variants in different normal and neoplastic tissues and cell lines varies significantly. The role of alternative splicing as the mechanism responsible for posttranscriptional regulation of mts1 gene expression is discussed.
Assuntos
Processamento Alternativo , Proteínas de Transporte/genética , Sequência de Bases , Proteínas de Transporte/metabolismo , Clonagem Molecular , Inibidor p16 de Quinase Dependente de Ciclina , Primers do DNA , DNA Complementar , Éxons , Humanos , Dados de Sequência Molecular , Processamento Pós-Transcricional do RNA , RNA Neoplásico/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The first intron of the mts1 gene, a gene which is selectively expressed in metastatic cells and in normal cells that are motile, was found to be highly homologous to the CD3 delta enhancer element. Because of the homology between the CD3 delta enhancer and the first intron of mts1, we analysed the first intron of the mts1 gene to determine whether it functions as a transcriptional regulatory element. Highly metastatic CSML-0 cells transfected with chloramphenicol acetyltransferase containing plasmids demonstrated the ability of the mts1 first intron to function as a positive regulatory element. In vitro footprinting analysis using extracts from CSML-0 cells (which express mts1 at low levels) of CSML-100 cells (which express mts1 at high levels) identified a protected 16 nucleotide element in the first intron of mts1, regardless of the extract used. However, in vivo footprinting analysis of the same region identified the protected 16 nucleotide fragment only in the mts1 intron from CSML-100 cells, not from CSML-0 cells. Differences in the methylation pattern of the mts1 gene in CSML-100 cells and CSML-0 cells are known to exist, and may in part be responsible for the mts1 footprinting differences observed in vivo from the different cells lines.
Assuntos
Íntrons , Metástase Neoplásica/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Complexo CD3/genética , Cloranfenicol O-Acetiltransferase/genética , DNA , Elementos Facilitadores Genéticos , Metilação , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Plasmídeos , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The mts1 gene is specifically expressed in metastatic tumors but not in non-metastatic counterparts. The gene was cloned from both mouse and human sources. The 5'-flanking regions of the mts1 gene from both sources were sequenced, revealing a 135 base pair region of high homology in the vicinity of the TATA box. The 5' region of the mts1 gene was also observed to have high degree of homology with some known promoter and enhancer sequences. The results of our transient transfection assays, in conjunction with those obtained from in vivo and in vitro footprinting of the promoter region, show no evidence of cis-control elements important for transcriptional regulation of mts1 gene. mts1 was found to be hypermethylated in CSML-0 cells and in the tissue types that do not express the gene or do that only at low levels. The possible role of methylation in progression of the nonmetastatic CSML-0 adenosarcoma cell line towards the metastatic CSML-100 adenosarcoma cell line will be discussed.
Assuntos
Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Elementos Facilitadores Genéticos/genética , Humanos , Metilação , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmídeos/genética , Homologia de Sequência do Ácido Nucleico , TATA Box , Transfecção/genética , Células Tumorais CultivadasRESUMO
A genomic copy of the mts271 gene which is specifically expressed in metastatic cells has been cloned and characterized. The gene consists of two exons and one intron and has an open-reading frame for the protein of 101 amino acids. The protein contains two helix-loop-helix calcium-binding domains, which is a common feature for the members of the large family of intracellular calcium-binding proteins (Ca B Ps). The primary structures of the mts271 gene products and other Ca B Ps were compared. High level of homology was found for S100 and calcium-binding protein of intestinal epithelium of rats. On the whole, the mts271 protein is a new calcium-binding protein which is specifically expressed in metastatic cells.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Metástase Neoplásica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Éxons , Íntrons , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Fenótipo , Plasmídeos , Ratos , Proteínas S100/genética , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Gene cloning was used with a view to examine differences in gene expression in metastatic (CSML-100) and non-metastatic (CSML-0) cell lines. Differential screening of the cDNA libraries obtained was performed. Mts271 gene which is specifically expressed in metastatic cells was isolated.
Assuntos
Clonagem Molecular , DNA de Neoplasias/isolamento & purificação , DNA/isolamento & purificação , Neoplasias Experimentais/genética , Transcrição Gênica , Animais , Southern Blotting , DNA/genética , DNA de Neoplasias/genética , Camundongos , Camundongos Endogâmicos A , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/patologia , Células Tumorais CultivadasRESUMO
Extrachromosomal closed circular IAP gene DNA was cloned from Ehrlich ascites carcinoma cells. Two clones were analysed in detain and the following conclusions were drawn: 1) there is closed circular IAP gene DNA in mouse cells; 2) the formation of circular DNA and the transposition of IAP gene by means of reverse transcriptase are documented.
Assuntos
Carcinoma de Ehrlich/genética , DNA Circular/genética , DNA de Neoplasias/genética , DNA Polimerase Dirigida por RNA/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Mapeamento Cromossômico , Clonagem Molecular , Elementos de DNA Transponíveis , Genes de Partícula A Intracisternal , Camundongos , Dados de Sequência MolecularRESUMO
A comparison of the expression of two mobile genetic elements A1 and B2 was studied in normal and tumor tissues. The A1 element is a chromosomal homolog of IAP genes, and B2 is a short ubiquitous repetitive sequences of the mouse genome. These sequences were earlier cloned in our laboratory and in this study were used as probes in hybridization experiments with RNA isolated from different mouse tumor and normal tissues. Both elements were efficiently transcribed in tumor cells. The level of expression of A1 sequences in tumors was 100-200 times higher than in normal tissues. The amount of B2 small cytoplasmic RNA significantly varied in different normal tissues. The content of this RNA was much higher in tumors. Closed circular DNA molecules containing IAP sequences were found in Ehrlich carcinoma cells. These DNA molecules are considered as intermediate forms of the mobile elements. The role of these mobile elements in the regulation of RNA expression and tumor progression is discussed.
